Project Summary E. coli is the most common gram-negative organism that causes neonatal meningitis and neonatal E. coli meningitis is associated with high mortality and morbidity. A major contributing factor to high mortality and morbidity is our inadequate knowledge on the pathogenesis of this disease. Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread, but it is incompletely understood how circulating E. coli traverses the blood-brain barrier. We have established both in vitro and in vivo models of the blood- brain barrier using human brain microvascular endothelial cells (HBMEC) and animal models of experimental hematogenous E. coli meningitis, respectively. Using these in vitro and in vivo models, we have shown that successful traversal of the blood-brain barrier by circulating E. coli K1 requires E. coli binding to and invasion of HBMEC, but the microbial-host interactions that are involved in E. coli binding to and invasion of HBMEC remain incompletely understood. During the previous funding period, we determined E. coli structures that contribute to HBMEC binding and invasion, and showed that those E. coli structures interact with their respective HBMEC receptors, demonstrating that E. coli binding to and invasion of HBMEC occurs as the result of ligand-receptor interactions. We also showed that those microbial-HBMEC interactions result in activation of specific host cell signal transduction pathways. In addition, we demonstrated for the first time that expression levels of the HBMEC receptors and modulation of host cell signaling molecules affect E. coli K1 invasion of HBMEC and penetration into the central nervous system (CNS). Our findings, thus, support the concept that down-modulation and/or blockade of microbial-host interactions involved in E. coli invasion of HBMEC provides a novel concept and approach to prevention of E. coli K1 traversal of the blood-brain barrier and penetration into the CNS, the essential step required for the development of E. coli meningitis. Based on these findings, we would like to continue investigating the E. coli K1-HBMEC interactions that affect E. coli invasion of HBMEC, the critical step for E. coli penetration into the CNS. Further understanding and characterization of the microbial-host interactions that are involved in E. coli invasion of HBMEC should enhance our understanding of the pathogenesis of E. coli meningitis and also help in developing a novel approach to prevention of meningitis, as exemplified in our progress report.